Article Betacoronavirus Adaptation to Humans Involved Progressive Loss of Hemagglutinin-Esterase Lectin Activity Graphical Abstract Highlights dAdaption of coronaviruses OC43 and HKU1 to humans involved loss of HE lectin function dOC43 HE receptor binding site was lost via progressive accumulation of mutations dLoss of HE receptor binding alters sialate-9-O-acetylesterase receptor destroying activity dBalance of receptor binding and receptor destruction contributes to host tropism Authors Mark J.G. Bakkers, Yifei Lang, Louris J. Feitsma, ., Martijn A. Langereis, Eric G. Huizinga, Raoul J. de Groot Correspondence r.j.degrootuu.nl In Brief Coronavirus OC43 entered the human population relatively recently. Bakkers et al. report that as an adaptation to replication in human airways, the OC43 hemagglutinin-esterase lost its receptor- binding function. Consequently, virion- associated receptor-destroying activity toward clustered sialoglycan-based receptor determinants was reduced. Suggestive of convergent evolution, human respiratory coronavirus HKU1 underwent similar changes. Accession Numbers 5N11 Animal beta-1 coronavirusesHuman coronavirus OC43 virion-associated receptor-destroying activity high reduced Zumla et al., 2015). However, four other respiratory coronavirusesalphacoronaviruses NL63 and 229E and lineage A betacoronaviruses OC43 and HKU1successfully breached the species barrier and are currently maintained in the human population worldwide through continuous circulation (Su et al., 2016; de Groot et al., 2011). Conceivably, the study of these genuine human coronaviruses (HCoVs) may yield clues to what is required for viral adaptation to the human host and thereby in- crease our understanding of the probabilities and risks of coro- navirus cross-species transmission. OC43 and HKU1, while generally associated with benign com- mon colds in healthy immunocompetent individuals, may cause signifi cant morbidity and even mortality in the frail (Morfopoulou et al., 2016; Woo et al., 2005a). OC43, the best-studied HCoV, apparently arose relatively recently, 120 to 70 years ago, with the most recent common ancestor of all extant OC43 variants dating to the 1950s (Lau et al., 2011; Vijgen et al., 2005, 2006). OC43groupsinthespeciesBetacoronavirus-1(b1CoV), together with highly related viruses from ruminants (bovine coro- navirus, BCoV), swine (porcine hemagglutinating encephalomy- elitis virus, PHEV), equines (equine coronavirus, ECoV), leporids (rabbit coronavirus HKU14, RbCoV), and canines (canine respi- ratory coronavirus, CRCoV) (de Groot et al., 2011; Figure 1A). The extraordinary radiation of b1CoVs might be explained from their receptor usage, as they attach to 9-O-acetylated sialic acids (9-O-Ac-Sias) (Matrosovich et al., 2015), i.e., glycan com- ponents common in mammals and birds (Traving and Schauer, 1998). Paradoxically, however, most b1CoVs, including OC43, have very narrow host ranges. They form distinct monophyletic clades congruent with host selectivity, and phylogenetic evi- dence strongly argues against recurrent inter-species transmis- sions (Lau et al., 2011; Vijgen et al., 2006; see also Figure 1A). With the emergence of OC43 seemingly sparked by a singular one-time zoonotic event, the founder virus likely possessed unique traits already to allow effi cient infection of, and transmis- sion among, humans. In turn, these traits and subsequent adaptations to the new niche must have closed the door on rein- troduction of the virus into animals. Despite the close genetic relationship between OC43 and BCoV-Mebus, particularly (96.6% identity across their entire genomes), their high-preva- lence endemicity/enzooticity, and the scale and frequency of interactions between their host species, there is no evidence of 356Cell Host de Groot et al., 2011; Hause et al., 2014; Matrosovich et al., 2015). HE monomers have a bimodular structure with a carbohydrate-binding (lectin) domain appended to an enzy- matically active sialate-O-acetylesterase (esterase) domain (Langereis et al., 2009; Rosenthal et al., 1998; Zeng et al., 2008). Typically, in b1CoVs, both the spikes and HEs bind 9-O-Ac-Sias, while the HE esterase domain promotes virus elution through receptor destruction. In consequence of the opposing activities of receptor binding and receptor destruction, b1CoV attachment to sialoglycans is dynamic and reversible. Thus, dead-end binding of virions to decoy receptors in oropha- ryngeal, respiratory, and gastrointestinal mucus may be pre- vented. Moreover, in infected host cells, HE-mediated receptor destruction is essential for effi cient release of viral progeny (Des- forges et al., 2013). Here we present a comprehensive structure-function study of OC43HE.WedemonstratethatoverdecadesafterOC43sintro- duction, its evolution was marked by a progressive loss of HE re- ceptor-binding activity through the accumulation of select muta- tions in the HE lectin domain. The effect of these mutations on the